Academic literature on the topic 'Conglutin'

The degradation of the major seed storage globulins of Lupinus albus L. was studied during the first 11 days after the onset of germination. The collagenolytic activity present in cotyledonary extracts was found to closely follow the rate and pattern of total globulin degradation. Little or no degradation of the three main globulins was observed during the first days of germination and seedling growth. The rate of proteolysis of the �-conglutin main subunit, composed of one heavy glycopolypeptide chain and one light non-glycosylated polypeptide chain, occurs gradually between days 3 and 11, with no apparent change in the globulin structure being detected during this time. Between days 3 and 5, �-conglutin, with at least 14 of its heavier polypeptide chains glycosylated, suffers a dramatic change in its structure and concentration, involving the appearance of a new set of polypeptides including a higher molecular mass group, whose concentration steadily declines until complete disappearance after 11 days, and a lighter molecular mass group, whose concentration surprisingly increases from 5 to 11 days. Finally, �-conglutin, with each of its four main subunits composed of a heavy glycopolypeptide chain and a 19 kDa non-glycosylated polypeptide chain, undergoes a pattern of degradation intermediate to those described for �- and �-conglutins in the sense that the new set of polypeptides coexist with polypeptides that were already present in the dry seed. The degradation of a-conglutin subunits starts at the level of their heavier polypeptide chains, releasing the intact 19 kDa polypeptide which gradually disappears after day 7. Our results indicate that �-, �- and �-conglutins are subjected to differential proteolysis during germination and seedling growth. Furthermore, the different polypeptide chains of each conglutin are also degraded at significantly different rates.

Narrow-leafed lupin (NLL; Lupinus angustifolius L.) has multiple nutraceutical properties that may result from unique structural features of β-conglutin proteins, such as the mobile arm at the N-terminal, a structural domain rich in α-helices. A similar domain has not been found in other vicilin proteins of legume species. We used affinity chromatography to purify recombinant complete and truncated (without the mobile arm domain, tβ5 and tβ7) forms of NLL β5 and β7 conglutin proteins. We then used biochemical and molecular biology techniques in ex vivo and in vitro systems to evaluate their anti-inflammatory activity and antioxidant capacity. The complete β5 and β7 conglutin proteins decreased pro-inflammatory mediator levels (e.g., nitric oxide), mRNA expression levels (iNOS, TNFα, IL-1β), and the protein levels of pro-inflammatory cytokine TNF-α, interleukins (IL-1β, IL-2, IL-6, IL-8, IL-12, IL-17, IL-27), and other mediators (INFγ, MOP, S-TNF-R1/-R2, and TWEAK), and exerted a regulatory oxidative balance effect in cells as demonstrated in glutathione, catalase, and superoxide dismutase assays. The truncated tβ5 and tβ7 conglutin proteins did not have these molecular effects. These results suggest that β5 and β7 conglutins have potential as functional food components due to their anti-inflammatory and oxidative cell state regulatory properties, and that the mobile arm of NLL β-conglutin proteins is a key domain in the development of nutraceutical properties, making NLL β5 and β7 excellent innovative candidates as functional foods.

Protein isolate obtained from sweet yellow lupin (Lupinus luteus L.) and its Alcalase hydrolysates were examined for their functional and antioxidant properties in relation to surface hydrophobicity of proteins and peptides and molecular weight distribution. Enzymatic hydrolysis improved the foaming characteristics of lupin proteins, while the emulsifying properties deteriorated. It means that good foaming properties of preparations are determined by the presence of low-molecular δ conglutin and small subunits of γ conglutins. In turn, larger proteins such as α and β conglutin are responsible for maintaining good emulsifying properties. The measured surface hydrophobicity was consistent with the results of emulsifying properties. It has also been shown that the scope of changes in antioxidant properties due to hydrolysis, measured by DPPH method and as reducing power, is more pronounced than with the use of ABTS and FRAP methods.

Agarose gel electrophoresis was used to separate three globular storage proteins from crude extracts of Lupinus albus seeds. Conglutin α, β, and γ purified by this technique were used as antigens to stimulate the production of antibodies in rabbits. The specificity of polyclonal antibodies produced was analysed by Western blot. Each antibody reacted with the specific antigen used to stimulate production of that antibody. In addition the anti-conglutin α preparation also showed binding to a region of the gel that comigrates with conglutin β. Key words: Lupin, conglutin, polyclonal antibody

The aim of the present study was to evaluate the effect of a chronic oral γ-conglutin treatment in male Sprague–Dawley rats in which hyperglycaemia had been induced by supplying 10 %d-glucose in drinking-water. A γ-conglutin dosage of 28 mg/kg body weight was daily administered to animals for 21 d. Plasma glucose, insulin and glucose overloading were monitored. Chronic administration of glucose resulted in a statistically significant (P < 0·05) increase in fasting blood glucose (2·5-fold) and insulin (2·7-fold)v.the values recorded in control rats. Simultaneous treatment with γ-conglutin attenuated the rise in plasma glucose (1·9-fold) and insulin (1·8-fold) levels in the glucose-fed rats (P < 0·05). Fasting insulin and homeostasis model of insulin resistance were decreased by 34 and 48 % (P < 0·05), respectively, in the γ-conglutin-treated ratsv.the values found in pair-fed animals. To confirm these results with a different approach, HepG2 cells, grown for 24 and 48 h in Dulbecco's minimum essential medium containing different glucose concentrations (5·5, 11·1 and 16·5 mmol/l), were exposed to 10 μmol/l γ-conglutin with or without 10 mmol/l metformin or 100 nmol/l insulin. γ-Conglutin increased glucose consumption (from 1·5- to 2·5-fold) in HepG2 cells, under all experimental conditions; this effect was more evident after 48 h incubation. Moreover, in thisin vitromodel, the addition of γ-conglutin potentiated the activity of insulin and metformin in cell glucose consumption. These findings extend the previous ones and suggest the potential use of lupin γ-conglutin in the control of glycaemia.

Proteins from the full and defatted flours of L. angustifolius cv Jurien and L. albus cv Murringo were prepared using alkaline extraction and iso-electric precipitation. Isolates were either freeze dried or spray dried or pasteurized at 75 ± 3 °C/5 min before freeze-drying. Various structural properties were investigated to elucidate the varietal and processing-induced effect on molecular and secondary structure. Irrespective of processing, isolated proteins had a similar molecular size, with α-conglutin (412 kDa) and β-conglutin (210 kDa) being principal fractions for the albus and angustifolius variety, respectively. Smaller peptide fragments were observed for the pasteurized and spray dried samples, indicating some degree of processing-induced changes. Furthermore, secondary structure characterization by Fourier-transform-infrared and circular dichroism spectroscopy showed β-sheet and α-helical structure being the dominant structure, respectively. Thermal characterization showed two denaturation peaks corresponding to β-conglutin (Td = 85–89 °C) and α-conglutin (Td = 102–105 °C) fractions. However, the enthalpy values for α-conglutin denaturation were significantly higher for albus species, which corroborates well with higher amounts of heat stable α-conglutin present. Amino acid profile was similar for all samples with limiting sulphur amino acid. In summary, commercial processing conditions did not have a profound effect on the various structural properties of lupin protein isolates, and properties were mainly determined by varietal differences.

γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteolytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated fromLupinus angustifoliusseeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and β subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographicD3symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a β-strand that is involved in intermolecular β-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.

The supplementation of different food items with grain legumes and, in particular, with lupin has been demonstrated to provide useful health benefits, especially in the area of cardiovascular disease prevention. In this work, label free quantitative untargeted and targeted approaches based on liquid chromatography−electrospray ionization−tandem mass spectrometry (LC−ESI−MS/MS) for investigating the protein profile of three pasta samples containing different percentages of narrow-leaf lupin flour were carried out. The untargeted method permitted the identification of the main acidic globulins (α-conglutin, β-conglutin, and δ-conglutin) and the comparison of their profile with raw lupin flour. The targeted method, based on High-performance liquid chromatography electrospray ionization tandem mass spectrometry HPLC-Chip-Multiple Reaction Monitoring (MRM) mode, allowed the quantification of γ-conglutin, the main hypoglycemic component of lupin protein: its concentration was around 2.25 mg/g in sample A, 2.16 mg/g in sample D, and 0.57 mg/g in sample F.

(1) Background: Inflammation molecular cues and insulin resistance development are some of the main contributors for the development and advance of the pathogenesis of inflammatory-related diseases; (2) Methods: We isolated and purified γ-conglutin protein from narrow-leafed lupin (NLL or blue lupin) mature seeds using affinity-chromatography to evaluate its anti-inflammatory activities at molecular level using both, a bacterial lipopolysaccharide (LPS)-induced inflammation and an insulin resistance pancreatic cell models; (3) Results: NLL γ-conglutin achieved a plethora of functional effects as the strong reduction of cell oxidative stress induced by inflammation through decreasing proteins carbonylation, nitric oxide synthesis and inducible nitric oxide synthase (iNOS) transcriptional levels, and raising glutathione (GSH) levels and modulation of superoxide dismutase (SOD) and catalase enzymes activities. γ-conglutin induced up-regulated transcriptomic and protein levels of insulin signalling pathway IRS-1, Glut-4, and PI3K, improving glucose uptake, while decreasing pro-inflammatory mediators as iNOs, TNFα, IL-1β, INFγ, IL-6, IL-12, IL-17, and IL-27; (4) Conclusion: These results suggest a promising use of NLL γ-conglutin protein in functional foods, which could also be implemented in alternative diagnosis and therapeutic molecular tools helping to prevent and treat inflammatory-related diseases.

gamma-Conglutin is a glycoprotein representing about 5% of lupin seed proteins. gamma-Conglutin is oligomeric at neutral pH, while at acidic pH it dissociates into monomers of 50 kDa. Each monomer is composed of two disulphide linked subunits of 17 and 29 kDa, originating from the endogenous proteolysis of the pro-polypeptide. Other 5 interchain disulphide bridges are also present. gamma-Conglutin sequence does not match any canonical legume storage proteins sequence; this protein is not degraded during seed germination and it was found to be extracellularly secreted during germination. These findings suggest a functional role for gamma-conglutin in the seed other than a mere storage one. gamma-Conglutin shows a great similarity (64%) with the basic globulin (BG7S) of soybean seed, which has been attributed an in vitro kinase activity and a binding activity towards some small regulatory proteins, such as a 4 kDa plant hormone-like peptide and mammalian insulin. -Conglutin shows resistance to in vitro proteolysis unless it was previously fully denatured. It can bind various divalent ions, such as Zn++ and Ni++, and protein such as acid phosphatase and insulin with a Kd of 10-5 M. Finally, gamma-conglutin was found to decrease glycemia in glucose overloading experiments on rats. Therefore, it seemed worth to investigate the molecular bases of this biological activity, as well as the properties related to gamma-conglutin metabolic fate and interaction with target cells. Indeed, aim of this work was to unravel the structural bases of gamma-conglutin assembly and stability to proteolysis and to develop suitable models to monitor its pathway after assumption and the cellular mechanisms involved in gamma-conglutin stimulation. By using dynamic light scattering, we investigated the assembly state of gamma-conglutin at different pH conditions. The results indicated a transition from monomer to oligomer, likely hexamer, via the transient formation of a dimer while pH was raised from 4.5 to neutral values. A model of these pH transitions based on the role of charge attraction/repulsions between acidic and basic amino acid residues in driving gamma-conglutin aggregation state was proposed. By using far- and near-UV circular dichroism, and intrinsic/ANS fluorescence analyses, the conformational changes as a function of pH were measured. All results suggested that the protein secondary and tertiary structures were unmodified by pH change above 3.5, while for lower values the structural collapse of the protein took place. Nevertheless, indirect evidence obtained with a set of proteolytic enzymes showed that gamma-conglutin became susceptible to proteolysis at pH values lower than 4.25, thus confirming its overall stability at neutral to slightly acidic pHs and suggesting also that a partial opening of the protein, without loss of overall three dimensional conformation, already occurred. Although γ-conglutin 3D structure is not available, it can be argued that its native conformation, which is stabilized also by a number of intrachain disulphide bonds, is extremely compact, especially if compared to the canonical seed storage proteins. The survival of intact gamma-conglutin into the intestinal lumen as well as its presence in the blood of suitable animal models were not proved with our assays in this work. However, the transit of gamma-conglutin through the intestinal barrier, by using in vitro and ex vivo models, namely Caco-2 cell monolayers and intestinal everted sacs, was demonstrated. Both experimental approaches showed that the transit through a transcellular way was possible for the intact gamma-conglutin, though limited to a small percentage of the applied protein. Although we were not able to detect gamma-conglutin in the sera samples of animals fed gamma-conglutin, the hypothesis of an at least partial preservation of the protein into the gastro-intestinal tract is also consistent with the observed immunogenic activity as measured in orally-treated mice in a parallel study. Eventually, we showed that the full length protein in its native conformation was capable to activate the insulin-signaling pathway in miocyte models in an insulin-like pattern, despite the dramatic structural differences between the two proteins. Moreover, the cellular absorption of gamma-conglutin by miocytes was shown by using a 2D electrophoretic approach. This finding, obtained with a cellular model not specifically involved in protein transport, confirmed the transcellular transport for gamma-conglutin observed with Caco2 cells. Very recent unpublished data reporting the results of the first human trial with healthy human subjects confirmed gamma-conglutin biological activity. Therefore, the present set of data is relevant to design further approaches aimed at exploiting lupin gamma-conglutin as a preventing and therapeutic agent in the cases of impaired glucose tolerance and possibly pre-diabetic and diabetic conditions.

Lupin has recently been added to the list of allergens requiring mandatory advisory labelling on foodstuffs sold in the European Union, and since December 2008 all products containing even trace amounts of lupin must be labelled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and “legumin-like”) and β-conglutin (7S and “vicilin-like”), and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. β-conglutin is the only conglutin currently included in the list of the International Union of Immunological Societies (IUIS), designated as Lup an 1. The overall objective of these PhD is the selection of aptamers that can detect this allergen. Nucleic acid aptamers are synthetic ligands selected from vast combinatorial libraries through a process referred to as SELEX – Systematic Evolution of Ligand By Exponential Enrichment. Aptamers possess unique chemical and biochemical characteristics, such as: well known chemistry and remarkable stability, moreover, aptamers can be selected against virtually any target and in non-physiological conditions. In order to achieve the overall objective, a set of subobjectives will be achieved. The first of these involves the elucidation of protocols for the selective extraction of each of the lupin α, β, γ, and δ subunits, resulting in (i) protocols that can be used for selective extraction and isolation of the lupin α, β, γ, and δ proteins from food for subsequent analysis; (ii) standards that can be used in analytical assays and tools; and (iii) target that can be used for the selection of aptamers specific to the β-conglutin subunit. The core of the work is the selection of aptamers against the allergen Lup an 1 using a SELEX procedure, as well the preparation of protocols that can be used to monitor the evolution of aptamer selection. The functionality of the aptamer is demonstratedby exploiting it in an enzyme linked oligonucleotide assay as well as apta-PCR. Finally the resulting aptamer candidates that exhibit high affinity are fully characterised, truncated, and the structure of the final truncated aptamer is elucidated

Lupin és una planta lleguminosa amb un alt valor nutricional utilitzada àmpliament a la regió mediterrània en els àpats diaris, aliments fermentats tradicionals, aliments al forn i salses, sense contenir gluten, podent ser el substitut de la soja. Lupin va ser afegit recentment a la llista d'al·lèrgens que requereixen l'etiquetatge obligatori d'assessorament sobre els productes alimentaris comercialitzats a la Unió Europea, i des de desembre de 2006, tots els productes que continguin fins i tot petites quantitats de tramús han d'estar correctament etiquetats. L'etiquetatge és d'alta importància ja que encara no hi ha un medicament que pugui prevenir les al·lèrgies alimentàries i l'única opció és evitar estrictament l'aliment que causa l'al·lèrgia. Les globulines que es troben en el tramús consisteixen en dos grans subunitats denominades α-conglutina i β-conglutina, i dos menors, γ-conglutina i δ-conglutina. β-conglutina és l'única conglutina actualment inclosa en la llista de la Unió Internacional de Societats d'Immunologia (UISI), designada com l’al·lergen anafilàctic Lup an 1. L'objectiu d'aquest treball és la detecció de l'al·lergen anafilàctic β-conglutina. Han estat descrits aspectes fonamentals com ara la selecció d'un aptàmer (β-CBA II) en contra de β-conglutina i l'avaluació de la seva afinitat i especificitat. Es van utilitzar tres metodologies diferents per a els estudis competitius i es va demostrar que l’aptàmer β-CBA II s’uneix a un lloc diferent de β-conglutina en comparació amb l’aptàmer mencionat anteriorment (β-CBA I). Finalment, el seleccionat aptàmer β-CBA II es va utilitzar per detectar β-conglutina desenvolupant-se una plataforma sensible, ràpida i fàcil d'usar, amb possibilitat d'adaptar-se al punt d'atenció.
Lupino es una planta leguminosa con un alto valor nutricional utilizada ampliamente en la región mediterránea en las comidas diarias, alimentos fermentados tradicionales, alimentos horneados y salsas, sin contener gluten, pudiendo ser el sustituto de la soja. Lupino fue añadido recientemente a la lista de alérgenos que requieren el etiquetado obligatorio de asesoramiento sobre los productos alimenticios comercializados en la Unión Europea, y desde diciembre de 2006, todos los productos que contengan incluso pequeñas cantidades de altramuz deben estar correctamente etiquetados. El etiquetado es de alta importancia ya que todavía no existe un medicamento que pueda prevenir las alergias alimentarias y la única opción es evitar estrictamente el alimento que causa la alergia. Las globulinas que se encuentran en el altramuz consisten en dos grandes subunidades denominadas α-conglutina y β-conglutina, y dos menores, γ-conglutina y δ-conglutina. β-conglutina es la única conglutina actualmente incluida en la lista de la Unión Internacional de Sociedades de Inmunología (UISI), designada como el alérgeno anafiláctico Lup an 1. El objetivo de este trabajo es la detección del alérgeno anafiláctico β-conglutina. Han sido descritos aspectos fundamentales tales como la selección de un aptámero (β-CBA II) en contra de β-conglutina y la evaluación de su afinidad y especificidad. Se utilizaron tres metodologías diferentes para los estudios competitivos y se demostró que el aptámero β-CBA II se une en un sitio diferente de β-conglutina en comparación con el aptámero mencionado anteriormente (β-CBA I). Finalmente, el seleccionado aptámero β-CBA II se usó para detectar β-conglutina desarrollándose una plataforma sensible, rápida y fácil de usar, con posibilidad de adaptarse en el punto de atención.
Lupin is a leguminous plant with a high nutritional value used widely in the Mediterranean region such as everyday meals, traditional fermented foods, baked foods and sauces with gluten-free properties and a chance for being the soy substitute. Lupin has recently been added to the list of allergens requiring mandatory advisory labelling on foodstuffs sold in the European Union, and since December 2006 all products containing even trace amounts of lupin must be labelled correctly. Labelling is high important since there is not yet a medication that can prevent food allergies and only strict avoidance of the allergy-causing food is the way to prevent a reaction. Lupin globulins consist of two major globulins termed α-conglutin and β-conglutin, and two minor globulins, γ-conglutin and δ-conglutin. β-conglutin is the only conglutin currently included in the list of the International Union of Immunological Societies (IUIS), designated as the anaphylactic Lup an 1 allergen. This work overviews the detection of the anaphylactic β-conglutin allergen. Fundamental aspects such as the selection of a second aptamer (β-CBA II) against β-conglutin and the evaluation of its affinity and specificity have been described. Three different methodologies were used for the competitive studies and it was demonstrated that the β-CBA II aptamer binds to the different aptatope of β-conglutin compared to the aptamer reported previously (β-CBA I). Finally, selected β-CBA II aptamer was used to detect β-conglutin using a sensitive, rapid and user-friendly platform, which can be easily adapted as point-of-care tests.

Bovine conglutinin is a member of the collectin family of innate immune system proteins. Crystals of the native protein have been grown and X-ray diffraction data collected. The space group is tetragonal P4) with one CRD per asymmetric unit. The crystallographic structure of a recombinant bovine conglutinin carbohydrate recognition domain (CRD) has been solved to 1.2SA in native and 1.46A in ligand-bound form. The native structure has been solved by molecular replacement with the coordinates of oneCRD of human trimeric surfactant protein-D (hSP-D) as the search" model. hSP-D is the closest molecule to conglutinin phylogenetically for which the crystallographic structure is known. The fold is demonstrated to be the same as the highly conserved C-type lectin fold found in coUectins. The ligand bound structure was solved by soaking N-acetylglucosamine, the highest affmity ligand, into native crystals. Binding is shown to be by vicinal equatorial hydroxyl groups on the sugar ring of the ligand, confirming the carbohydrate recognition previously demonstrated for hSP-D and rat mannose binding proteins A and C. An additional hydrogen bond is observed between the acetyl group of the ligand and a protein lysine residue which could account for the high affinity of conglutinin for this ligand. The fmal R-factors are Rc:onv 21.7, Rrn,., 24.3 for the native, and ~v 20.3, Rne 22.3 for the ligand-bound structure. Attempts were made to grow crystals of a trimeric conglutinin fragment consisting of the a-helical coiled-coil neck region and three eROs. The crystals grown did not diffract to high enough resolution to allow data collection and structure solution. Reports that N-acetylmuramic acid, a component of bacterial cell wall peptidoglycan, was a high affinity ligand for hSP-D were followed up. Cocrystailisation trials failed to grow diffraction quality crystals.

Recognition of sugar moieties on the surface of microorganisms is one of the ways the body distinguishes potential pathogens from self-cells. The sugarbinding proteins, lectins, mediate this recognition role of the first line of defence against infections, preceding the antibody-mediated (adaptive) immune response. Collectins are calcium-dependent carbohydrate-binding proteins that have been implicated in innate immunity. Bovine conglutinin (BC) and lung surfactant protein-D (SP-D), belong to the family of 'collectins' which are characterised by four domains: an N-terminal cysteine-rich region, a collagenlike region linked with the carbohydrate recognition domain (CRD) via an ahelical neck region. BC and SP-D show remarkable similarity in their amino acid sequence (79% identity), function and biological characteristics. They have been shown to mediate microbial clearance either by directly binding to bacteria leading to phagocytosis or interacting with complement system components. The present study aims to elucidate the biological function of these proteins more precisely. Recombinant fragments (r) of BC and SP-D consisting of their CRDs and neck regions have been cloned in pET-21a and pMal-c2 vectors respectively, for expression in Escherichia coli. Recombinant conglutinin was expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturing procedure. Binding of rBC(N/CRD) to mannan and complement component, iC3b, was assessed in real-time by BIAcore. The dissociation constants were calculated by Scatchard analysis. The carbohydrate structures present on the surface of the microorganisms play an important role in mediating the interactions with the immune cells. The recombinant molecules showed calcium-dependent binding to lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited in presence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed by ELISA and retained its capacity to recognise various complement system components and the carbohydrate moieties on the surface of various pathogenic microorganisms. The recombinant protein retained its ability to bind various sugar residues, although with lower affinity than that of the native molecule. rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination of bacterial cell suspensions. A novel model has been used to describe the interactions of the collectins at the molecular level based on specificity of carbohydrate-recognition by the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeae has sequential deletions of the terminal sugars in their lipooligosaccharides (LOS). Conglutinin showed a preferential high affinity binding to 1291a mutant that expresses GlcNAc as the terminal hexose, in comparison to other mutants. This provides a unique system to understand the specific cell-surface interactions in relevance to a particular lectin. Further elucidation of the function of CRD and neck region at a structural level is in progress, using X-ray crystallography. Since the submission of the thesis, the structure of the monomeric CRD has been solved, which revealed a remarkable similarity to the SP-D and MBL structure. Trials are underway to get the structure of the trimeric CRDs. These studies aim to provide a better understanding of the collectinpathogen interaction at the biological and structural levels. The ultimate aim is to determine if the recombinant forms of these proteins can be used therapeutically to enhance the uptake and killing of pathogens.

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2015
Os fungos patogénicos representam, à escala mundial, uma séria ameaça para a saúde humana, tendo-se vindo a registar um aumento significativo de infeções fúngicas devido a fungos patogénicos oportunistas, o que as torna num fator importante de morbidade e mortalidade. Apesar de Candida albicans continuar a liderar as espécies de Candida causadoras de infeções da corrente sanguínea (ICS), nas últimas duas décadas tem ocorrido um desvio epidemiológico para um aumento de espécies de Candida "não-albicans". Infeções causadas por outras espécies de Candida têm vindo a aumentar, sendo responsáveis por mais de 90% das infeções fúngicas invasivas. De entre essas espécies, apenas C. glabrata pode ser verdadeiramente reconhecida como uma espécie emergente causadora de ICS, devido à sua resistência intrínseca e adquirida aos azóis e a outros agentes antifúngicos. Nenhuma das classes de antifúngicos atualmente disponíveis possui todas as características ideais de um agente antifúngico, o que, em última análise, conduz a falhas no tratamento. Como consequência, novas formulações de antifúngicos, combinações terapêuticas e o desenvolvimento de novos compostos bioativos podem ser úteis na obtenção de melhores resultados terapêuticos. As sementes de leguminosas são fontes bastante abundantes de proteínas, sendo o género Lupinus um dos mais ricos neste tipo de proteínas de reserva. As proteínas de reserva das sementes de leguminosas, classificadas como globulinas, compreendem duas famílias principais de proteínas: as vicilinas e as leguminas, que constituem aproximadamente 80% das proteínas totais das suas sementes, atuando não só como reservas de nutrientes durante a germinação, como também como proteínas de defesa para as plantas. A β-conglutina é a principal proteína de reserva nas sementes das espécies de Lupinus, sendo as suas subunidades sintetizadas durante o desenvolvimento cotiledonar de L. albus, a partir de um único polipéptido precursor glicosilado. Após a germinação, as subunidades da β-conglutina sofrem uma grande alteração na sua estrutura e concentração, levando ao aparecimento de um novo conjunto de polipéptidos. Recentemente, foi acidentalmente descoberto no nosso laboratório que a β-conglutina purificada de L. albus sofre, in vitro, um processo de proteólise. Após uma incubação de 7 dias a 25ºC, a β-conglutina sofre uma degradação controlada originando um oligómero estável e com atividade antifúngica, que denominámos PDβ. Tendo em conta o conhecimento prévio da atividade antifúngica do PDβ, este plano de trabalho teve como objetivos principais caracterizar o oligómero e determinar a sua estabilidade e a sua atividade antifúngica, bem como tentar perceber o seu mecanismo de ação como agente antifúngico, avaliando os efeitos fisiológicos e morfológicos em C. glabrata. A primeira fase do trabalho passou por isolar as globulinas totais dos cotilédones de L. albus seguida de purificação da fração correspondente à β-conglutina. Após purificação, a β-conglutina foi incubada a 25°C durante 7 dias, de modo a ocorrer a degradação para PDβ. Começou por se tentar perceber se o resultado desta degradação é devido a uma destruição da estrutura original da proteína dando origem a subunidades independentes. Os resultados permitiram concluir que embora a proteína sofra um processo de degradação, a sua massa molecular aumenta ligeiramente, o que permite concluir que o processo in vitro não se traduz num catabolismo com desaparecimento de sub-unidades mas sim de um arranjo estrutural diferente. O passo seguinte passou por avaliar a evolução do perfil polipeptídico e da atividade antifúngica durante o processo de degradação. Foi possível determinar que a degradação da β-conglutina começa a ocorrer a partir do terceiro dia de incubação a 25°C, sendo o pico máximo de degradação ao fim dos 7 dias. Esta degradação leva a uma acumulação progressiva de um polipéptido com 20 kDa, o que parece semelhante ao catabolismo que ocorre naturalmente nos cotilédones de L. albus. No entanto no processo in vivo a degradação origina exclusivamente um polipéptido de 20 kDa denominado Blad, que acaba por ser completamente degradado. Neste caso, o processo é interrompido sugerindo que falta uma peça chave para posterior degradação. Em relação à atividade antifúngica, o seu aparecimento é gradual: vai aumentando atingindo o pico máximo de atividade aos 7 dias de incubação. De seguida tentou caracterizar-se e identificar o mecanismo envolvido na degradação da β-conglutina em PDβ, começando por se analisar se poderia ser causada por uma reação de oxidação-redução, interferindo com o oxigénio disponível na reação. Os resultados obtidos indicam que o aumento de oxigénio disponível na reação não tem qualquer influência sobre a reação de degradação mas quando se adiciona dithiothreitol (DTT) a degradação não ocorre, ou é interrompida no momento em que este é adicionado. Uma possível explicação pode ser pelo facto de o DTT ser um eficaz desnaturante de proteínas quebrando as ligações dissulfídicas nos grupos de cisteína. Tendo em conta estes resultados e admitindo a hipótese de haver uma protease envolvida no processo de conversão a qual poderia estar a ser inibida por um agente redutor, foram testados vários inibidores de diferentes classes de proteases, adicionando-os separadamente à β-conglutina antes do período de incubação in vitro de 7 dias. Dos inibidores de proteases testados, apenas o ácido etilenodiaminatetraacético (EDTA) teve influência na β-conglutina, impedindo a sua degradação. De acordo com este resultado é possível concluir que a degradação da β-conglutina é dependente de uma metaloprotease, inibida aquando da adição de EDTA, resultado confirmado por recurso a uma zimografia. Esta metaloprotease encontra-se sob a forma inativa, encontrando-se apenas ativa ao fim de 3 dias de incubação da β-conglutina a 25°C, altura em que se inicia o processo de degradação. Para esclarecer a origem da molécula responsável pela degradação da β-conglutina, levantou-se a hipótese de se tratar de uma contaminação externa. No entanto, esta hipótese foi abandonada uma vez que tanto a solução de β-conglutina filtrada através de filtros de fluoreto de polivinilideno (PVDF) como a β-conglutina submetida a um tratamento com antibiótico sofreram o processo normal de degradação. Embora nesta fase do trabalho existissem resultados bastante interessantes, nenhum permitiu identificar a molécula responsável pela degradação da β-conglutina. Uma vez que os ensaios de zimografia foram os que permitiram chegar mais perto da sua identidade, foi efetuada uma nova zimografia utilizando as vicilinas totais como substrato, visto ser o substrato natural da metaloprotease em questão. As bandas com atividade proteolítica foram excisadas do gel e enviadas para sequenciação. No entanto, após receção dos resultados verificou-se que não foi possível identificar a possível metaloprotease. Em simultâneo com a caracterização bioquímica do processo de degradação, foi avaliada a atividade antimicrobiana de PDβ, o que revelou que apesar de PDβ não apresentar atividade bactericida, esta apresenta atividade fungicida em várias espécies de leveduras e de fungos filamentosos. Após o rastreio efetuado, foram avaliados os efeitos morfológicos e fisiológicos da PDβ, usando C. glabrata ISA 2163 como modelo. Começou por se comparar a actividade antifúngica de PDβ com a do itraconazole e anfotericina B, o que revelou que PDβ parece apresentar maior atividade inibitória e fungicida numa base molecular do que tanto o itraconazole como a anfotericina B. De seguida, realizaram-se curvas de crescimento de C. glabrata ISA 2163 exposta às concentrações inibitórias e letais de PDβ e de anfotericina B, de modo a estudar e comparar os seus efeitos no crescimento da levedura. As curvas obtidas permitiram concluir que a adição tanto de PDβ como de anfotericina B teve um forte efeito inibitório no crescimento de C. glabrata ISA 2163, visto que as células expostas a ambas as concentrações testadas de ambas as drogas apresentam uma diminuição na taxa de crescimento, quando comparado com a situação controlo (sem droga). Em ambos os casos, os efeitos de inibição começam a ocorrer ao fim de 4 h de incubação, verificando-se uma estabilização da DO640nm e das contagens de unidades formadoras de colónias (UFC), mais acentuada a partir das 12 h de incubação, na fração incubada com a concentração letal de PDβ. Simultaneamente foi avaliado o efeito de PDβ na atividade metabólica, com recurso ao fluorocromo FUN-1, e na integridade da parede celular, com recurso ao calcofluor white, de C. glabrata ISA2163, recolhendo amostras ao longo da curva de crescimento. Os resultados obtidos sugerem que, mesmo ao fim de 24 h de incubação com uma concentração letal de PDβ, não ocorre perda de viabilidade celular sugerindo que as células apenas perdem a capacidade de se multiplicar, dada a estabilização do número de células. Relativamente à integridade da parede celular, existe uma diferença notória entre a fração incubada com PDβ e a fração controlo. Enquanto a integridade da parede celular da fração controlo permanece inalterada ao longo da curva, a fração exposta à PDβ apresenta falhas na marcação com calcofluor white, a partir das 8 h de incubação, tornando-se mais evidentes após as 12 h. Estes resultados sugerem perda de integridade da parede celular e podem ser a razão pela qual as células perdem a capacidade para se multiplicar. Por último, determinou-se a localização celular da PDβ ao fim de 24 h de incubação em C. glabrata ISA 2163. Os resultados obtidos sugerem que PDβ se liga à parede celular da levedura, destabilizando-a. No entanto, não há vestígios da proteína no interior da célula. Este trabalho permite concluir que a degradação in vitro da β-conglutina para PDβ ocorre de forma controlada, possivelmente sob a ação de uma metaloprotease. A atividade desta metaloprotease só é visível após o início da conversão da β-conglutina, aumentando de atividade à medida que o oligómero é convertido. Isto sugere que na semente seca, quando se purifica a β-conglutina, a metaloprotease se encontra inativa, sendo necessária a sua ativação para que a conversão da β-conglutina tenha início. No que respeita a atividade antimicrobiana de PDβ, é possível concluir que a mesma apresenta uma forte atividade em diferentes fungos, tanto leveduras, patogénicas e alimentares, como fungos filamentos. Relativamente ao modo de ação do oligómero, verificou-se que este tem capacidade para se ligar à parede celular da levedura em estudo, criando danos significativos e afetando a sua integridade celular. Os danos causados ao nível da parede celular parecem ser suficientes para impedir a multiplicação do microrganismo, levando à inibição do seu crescimento.
Pathogenic fungi represent, worldwide, a serious threat for human’s health. Candida species are among the top ten pathogens causing bloodstream infections (BSI). C. glabrata can be described as a truly emerging pathogen that cause BSI due, in part, to its intrinsic and acquired resistance to azoles and other commonly used antifungal agents. All antifungal agents available display several disadvantages. It is therefore essential to identify new, potent and safe antifungal drugs with novel modes of action. PDβ is an in vitro breakdown oligomer resultant from the degradation of β-conglutin, with proved antifungal activity. Based on this information and in an attempt to go deeper in knowledge, the first objective of this work was the biochemical characterization of PDβ oligomer and the understanding of the metabolic route involved in the degradation process. This process was found to occur gradually and controlled by a metalloproteinase that needs to be activated, converting an inactive protein into an oligomer with antifungal activity. The second goal was the characterization of PDβ stability and antimicrobial activity against an array of different species. The antimicrobial activity of PDβ was shown to be quite diverse among yeasts and filamentous fungi; however no bactericidal activity was detected. Finally, an attempt to understand PDβ mode of action by assessing its physiological and morphological effects on C. glabrata was explored. Upon exposure of C. glabrata to PDβ, the yeast is still metabolically active but loses its cell wall integrity. Furthermore, the activity of PDβ suggests binding to the cell wall, without entering into the cell. The binding of PDβ leads to damages and destabilization of the cell wall which are severe enough to prevent C. glabrata ISA 2163 multiplication.